To characterize functions of the HIV-1 Gag matrix (MA) protein and define domains involved in these functions, we have introduced over 70 single and double amino acid mutations throughout MA. Biological and biochemical analysis identified classes of mutations which: i) impaired Gag membrane binding or myristylation, ii) blocked Env incorporation into virions, iii) blocked virus assembly, iv) impaired virus entry, or v) redirected assembly from the plasma membrane to cytoplasmic compartments. During the course of these studies, viral revertants of all classes of mutations were obtained and characterized. The majority of second-site changes identified in the MA region of these revertant viruses were located distantly from the original mutations in the primary sequence. When introduced into MA in the presence of the original mutations, these second-site changes compensated for the defects caused by the original substitutions, suggesting a direct or functional interaction between the pairs of distantly-located amino acids. The second-site changes were also introduced in the context of wild-type MA and some of them caused defects in HIV-1 replication, suggesting structural or functional interdependency between the second-site and original mutations. The mutational analysis and characterization of revertants further our understanding of the relationship between MA structure and function, and enable us to delineate the role of MA in the HIV-1 life cycle. Sequence comparison of a number of primate lentivirus isolates reveals a very high degree of sequence conservation at MA amino acid 20 (Leu). Mutations at this position, in particular Lys and Arg substitutions, delayed or blocked virus replication in T-cell lines, primary PBMC, and human macrophages. Biochemical analyses revealed that the residue 20 mutations did not affect virus assembly and release, Env incorporation into virions, exogenous RT activity, or RNA encapsidation. However, a defect early in the virus life cycle was observed in single cycle (MAGI) infectivity assays, which demonstrated a 10-fold reduction in virus infectivity. Kinetic analyses of reverse transcription by PCR using a range of primers at early time points post-infection in both primary macrophages and T-cell lines indicated that viral DNA synthesis was impaired in cells infected with the mutant virus. Intriguingly, viral DNA synthesis was diminished in endogenous RT assays. A viral revertant maintained the position 20 change and acquired second-site changes at residues 73 and 82. Interestingly, activity of the revertant virus in the endogenous RT assay was restored to wild-type levels. Lentiviruses, including HIV-1, possess transmembrane envelope (Env) glycoproteins with unusually long cytoplasmic tails. A prominent structural feature of the HIV-1 cytoplasmic domain is the presence of two predicted a-helices in the central (a-helix 2) and C-terminal (a-helix 1) portions of the cytoplasmic domain. To characterize these domains, we have introduced a number of truncations, deletions, and single and double amino acid substitution mutations throughout both a-helical domains. The effects on Env expression, Env incorporation into virions, virus infectivity in MAGI cells, virus replication in T-cells, and syncytium formation have been assessed. The results indicate that the gp41 cytoplasmic tail a-helical domains play an important role in Env incorporation and virus infectivity, and identify specific functional determinants within these domains. We also observed that several mutations in gp41 a-helix 2 markedly impair Gag processing, and that this defective Gag processing is relieved by a matrix (MA) mutation which blocks Env incorporation into virions. This phenomenon supports the hypothesis that MA and gp41 interact during Gag and Env transport and virus assembly. It has been suggested that the matrix (MA) domain of HIV-1 Gag plays a central role in translocating the HIV-1 preintegration complex to the nucleus following infection of non-dividing cells. Recent publications proposed that phosphorylation of 1% of MA on a C-terminal Tyr (residue 131) was required to reverse the membrane binding properties of MA and promote an association between MA and the integrase protein, thus enabling MA to assist in translocating the HIV-1 preintegration complex to the nucleus. The critical piece of biological data in support of this model was that mutation of MA Tyr 131 to Phe blocked infection of differentiated primary human monocyte-derived macrophages. To confirm this observation, we introduced the same single amino acid substitution (Tyr->Phe) at residue 131 of MA. We demonstrated that: i) in cells from numerous macrophage donors, ii) in two different primary macrophage purification/culture systems, iii) in the context of two different macrophage-tropic molecular clones, and iv) in the presence or absence of a functional vpr gene, the 131YF mutation had no detectable effect on infectivity. These data, together with our previous studies, challenge the hypothesis that MA is involved in nuclear targeting of the viral preintegration complex in terminally differentiated primary human macrophages.